Oral Abundance of Actinomyces spp. in Breast Cancer Patients.

OBJECTIVES: Pathophysiology of medication-related osteonecrosis of the jaw (MRONJ) is still unclear, and disease development is associated with adverse reaction of bisphosphonates and denosumab, and Actinomyces spp. as well. In this study, we evaluated the abundance of Actinomyces spp. in breast cancer patients undergoing chemotherapy compared to healthy controls. METHODS: Oropharyngeal samples were collected from treatment-naive early-stage breast cancer patients, who were scheduled for standard of care therapy (eight samples throughout chemotherapy, one prior to radiotherapy and one after a year of start), as well as from healthy controls at matched timepoints. We quantified Actinomyces spp. in the samples with a highly sensitive and specific quantitative polymerase chain reaction. RESULTS: Twenty-one patients and 16 healthy subjects were enrolled. Forty-eight percent of patients suffered from estrogen receptor-positive/progesterone receptor-positive or -negative/human epidermal growth factor receptor 2 (HER2)-negative disease, 38% were HER2-positive, and 14% were triple-negative. Comparison of Actinomyces spp. loads in cancer patients and healthy controls did not reveal significant difference. Fluctuations on bacterial quantity were observed in both groups over time. Tumor receptor status or different chemotherapy schemes of patients were not correlated with a particular pattern on abundance of Actinomyces spp. CONCLUSIONS: We suggest that Actinomyces spp. are not the initiative factors in MRONJ development. These bacteria are not altered in abundance during chemotherapy, but they behave opportunistic when there is a bone disruption in the oropharynx in the first place caused by antiresorptive drugs or dental trauma and proliferate in their new niche. Thus, Actinomyces spp. plays a latter role in MRONJ development, rather than a primary causative one.

Tooth extraction in mice administered zoledronate increases inflammatory cytokine levels and promotes osteonecrosis of the jaw.

INTRODUCTION: Osteonecrosis of the jaw (ONJ) occurring after invasive dental treatment often adversely affects patients' activities of daily living. Long-term administration of strong anti-bone resorptive agents such as bisphosphonates prior to invasive dental treatment is considered an ONJ risk factor; however, pathological mechanisms underlying ONJ development remain unclear. MATERIALS AND METHODS: We developed an ONJ mouse model in which a tooth is extracted during treatment with the bisphosphonate zoledronate. RESULTS: We observed induction of apoptosis in osteocytes, resulting in formation of empty lacunae in jaw bones at sites of tooth extraction but not in other bones of the same mice. We also observed elevated levels of inflammatory cytokines such as TNFα, IL-6 and IL-1 in jaw bone at the extraction site relative to other sites in zoledronate-treated mice. We also report that treatment in vitro with either zoledronate or an extract from Porphyromonas gingivalis, an oral bacteria, promotes expression of inflammatory cytokines in osteoclast progenitor cells. We demonstrate that gene-targeting of either TNFα, IL-6 or IL-1 or treatment with etanercept, a TNFα inhibitor, or a neutralizing antibody against IL-6 can antagonize ONJ development caused by combined tooth extraction and zoledronate treatment. CONCLUSIONS: Taken together, the cytokine storm induced by invasive dental treatment under bisphosphonate treatment promotes ONJ development due to elevated levels of inflammatory cytokine-producing cells. Our work identifies novel targets potentially useful to prevent ONJ.

Comparison of the histopathological characteristics of osteomyelitis, medication-related osteonecrosis of the jaw, and osteoradionecrosis.

The purpose of this study was to compare the histopathological parameters of chronic/suppurative osteomyelitis, medication-related osteonecrosis of the jaw (MRONJ), and osteoradionecrosis (ORN), and to examine the hypothesis that distinct histological features can be related to a specific disease, allowing for diagnosis based on microscopic evaluation alone. One hundred and ten samples were reviewed by two examiners in a blinded fashion, and a semi-quantitative histomorphometric analysis was performed. The parameters evaluated included the presence or absence of necrotic bone, inflammation, reactive bone formation, bacteria, and osteoclasts. No statistically significant differences were found between groups for any parameter. Necrotic bone was common to all three diagnoses. Inflammation and reactive bone formation were present in all three diagnoses. The presence of bacteria was a prominent feature in all cases. Osteoclasts were scarce in MRONJ and osteomyelitis, and non-existent in ORN. The results of this study failed to identify distinctive microscopic characteristics in any of the three entities that could be used to differentiate between them. Therefore, it is impossible to reach a specific final diagnosis based on microscopic findings alone. The role of microscopic analysis is to serve as an aid to diagnosis that must be complemented by the patient's history and imaging.

Microbial diversity in infections of patients with medication-related osteonecrosis of the jaw.

OBJECTIVES: A central role of infections in the treatment of MRONJ patients is widely accepted. An investigation of the MRONJ lesions' biofilms as potential pathogens seems logical. MATERIALS AND METHODS: We investigated the clinical data of our MRONJ patients who received surgery in advanced stage of the disease. Special attention was granted to the local colonizers harvested from osseous MRONJ specimens and submucosal putrid infections. RESULTS: Eleven out of 71 patients presented a spontaneous onset of the disease and for 60 out of 71 patients a trigger was detected. Breast cancer (29.6%) and prostate cancer (22.5%) were the most frequent underlying disease for prescription of an antiresorptive therapy, mostly zoledronate. Submucosal soft tissue biofilms significantly differed from biofilms harvested from the MRONJ lesions bottom, yet the most frequent bacteria were equally present in both groups: Streptococcus species (spp.), Prevotella spp., Actinomyces spp., Veillonella spp., and Parvimonas micra. The cephalosporins, cefuroxime and cefotaxime, and ß-lactam antibiotics with ß-lactamase inhibitor revealed the greatest susceptibility for the detected bacteria. CONCLUSION: The bacteria from the submucosal areas and the bottom of the infected bone presented comparable susceptibility to the common antibiotics regimes. Streptococcus spp., Prevotella spp., and Veillonella spp. present a high abundance in MRONJ lesions beside Actinomyces spp. The MRONJ lesions bottom is in many cases not infected by Actinomyces spp. CLINICAL RELEVANCE: The removal of the necrotic bone reduces the variety of bacteria found in MRONJ lesions, in particular at the bottom of the lesion.

Microbial population changes in patients with medication-related osteonecrosis of the jaw treated with systemic antibiotics.

OBJECTIVE: This study aimed to investigate the bacterial population in patients with medication-related osteonecrosis of the jaw (MRONJ) after treatment with doxycycline and metronidazole. STUDY DESIGN: A total of 38 patients with MRONJ (age range 55-88, mean age 73 + 8.82 standard deviation) treated with doxycycline first and with metronidazole second were enrolled in this study. Two swabs were taken at the margin of the infected MRONJ lesion after applying pressure on the marginal mucosa, and visible pus was secreted. Real-time polymerase chain reaction was used to analyze 20 periopathogenic and commensal species and the total bacterial level. Bacterial counts were compared between antibiotic treatments and with a control group of orally healthy patients who didn't have periodontal pockets of more than 3 mm (n = 29) by means of a Mann-Whitney U test. Comparisons between the two antibiotic treatments were performed by a paired Wilcoxon signed rank test. RESULTS: The total bacterial level was significantly higher in the MRONJ patients treated with systemic antibiotics compared with the control group. However, significant lower bacterial amounts were found for 12 of the 20 investigated bacteria. We couldn't establish a significant advantage of metronidazole administration after doxycycline treatment. CONCLUSION: Our findings suggest that the total bacterial level in MRONJ patients is higher even when treated with systemic antibiotics. The significantly different bacterial amounts of the selected species suggest an alteration in the microbial population.

Role of microbiological culture and polymerase chain reaction (PCR) of actinomyces in medication-related osteonecrosis of the jaw (MRONJ).

We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new approaches for the rapid detection of microorganisms including those difficult to culture. The aim of our study is to identify the bacterial profiles in MRONJ by microbiological culture and polymerase chain reactions (PCR). A retrospective analysis was performed on MRONJ patients from 2008 to 2014. The bacterial profile from MRONJ bone samples was determined using microbiological culture and PCR. Ninety five patients fulfilled the inclusion criteria with mean age of 69.85 ± 8.71 years. A female predilection was detected. The mandible was more commonly affected than maxilla. Tooth extraction was the frequent triggering factor. Breast cancer was the primary cause for administration and intravenous bisphosphonates were the most commonly administrated antiresorptive drugs. The majority of patients were classified as stage 2. Posterior teeth were most commonly affected. Based on bone culture results, the most common microorganism were both actinomyces and mixed flora. PCR confirmed the presence of actinomyces in 55 patients. Our data suggest that PCR might be an innovative method for detection of microorganisms difficult to culture using traditional microbiological techniques.

Bacterial diversity in medication-related osteonecrosis of the jaw.

OBJECTIVE: The aim was to study the association between microflora and medication-related osteonecrosis of the jaw (MRONJ) by using culture-independent molecular techniques to detect bacteria in necrotic bone lesions. STUDY DESIGN: Included were 18 consecutive patients with MRONJ, 10 with osteoporosis and 8 cancer patients. Bone biopsies were retrieved from the center of the necrotic bone and from visually healthy bone, and 16 S rRNA gene fragments from bacterial DNA were amplified with polymerase chain reaction. RESULTS: The study revealed a diversity of bacteria represented by 16 S rRNA sequences in all the necrotic bone samples and in 60% of the visually healthy bone. Eight dominating taxa groups were identified at the genus level: Porphyromonas, Lactobacillus, Tannerella, Prevotella, Actinomyces, Treponema, Streptococcus, and Fusobacterium. CONCLUSIONS: The necrotic bone lesions contained mainly anaerobic bacteria, representative of periodontal microflora, suggesting that a periodontal infection in combination with antiresorptive treatment could initiate osteonecrosis.

Profile of bacteria colonizing the exposed bone of patients with anti-osteoclastic drug-related osteonecrosis of the jaws.

Microbial etiology for anti-osteoclastic drug-related osteonecrosis of the jaw (ARONJ) was suggested. This study investigates any link between bacteria colonizing ARONJ sites and other oral cavity sites. Microbiota samples of 10 ARONJ patients were collected from the exposed bone, adjacent teeth, contralateral teeth, and tongue. DNA checkerboard hybridization was used for microbiota analysis with 43 genomic DNA probes prepared from human oral bacterial (38) and candida (5) species, using Socransky's bacterial complexes as a guide. The frequency and the mean proportion of each bacterial species were used. Eikenella corrodens, Streptococcus constellatus, and Fusobacterium nucleatum were dominant in the ARONJ sites and detected in most teeth samples. Staphylococcus aureus was also dominant in the ARONJ sites and tongue. Significant correlations were found between the mean proportions of bacterial species colonizing adjacent teeth, contralateral teeth, and tongue (p < 0.001, R(2) > 0.69). No significant correlation (p > 0.05, R(2) < 0.025) was found between bacteria colonizing ARONJ sites and other evaluated sites. Within the study limitations, it was concluded that the primary sources of microorganisms colonizing ARONJ sites could be other sites such as teeth and tongue. The microbial profile of the necrotic bone is predominantly colonized with bacteria from Socransky's green and orange complexes, as well as with species associated with bone infections.

[Conditionally pathogenic microorganisms in patients with bisphosphonate jaw osteonecrosis].

The objective of the study was to define treatment strategy in cases of facial bones bisphosphonate induced osteonecrosis based on the study of the role of conditionally pathogenic oral microorganisms. Three typical clinical cases of bisphosphonate osteonecrosis of the facial bones were analyzed and 15 conditionally pathogenic oral microorganisms were identified in these patients using real-time PCR in saliva, wound and bone samples. A comparative analysis was carried out with purulent-inflammatory diseases of maxillofacial area. The study results proved an important role of conditionally pathogenic microorganisms of the oral cavity in the development of osteonecrosis of the facial bones. Wide range of bacterial species was identified in osteonecrosis of the facial bones patients. While bone tissue is most exposed to microbial communities, surgical treatment results in effective rehabilitation for a long period.

Bacterial profile and bone healing in rats receiving cancer therapeutic doses of bisphosphonates and corticosteroids: a pilot study.

The microbial aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains undefined. This study investigated the oral microbiota and socket healing after zoledronic acid (ZA) and dexamethasone (DX) administration. Fourteen rats assigned randomly to experimental (n=8) and control (n=6) groups were injected with ZA+DX or saline, respectively, for 3 weeks prior to and 9 weeks after the extraction of left first upper and lower molars. Whole genomic DNA probes of 38 bacterial species and five Candida species were hybridized to DNA extracted from biofilm samples on exposed bone and adjacent teeth. Only experimental rats exhibited exposed bone at euthanasia. All BRONJ-like lesions were colonized by Staphylococcus pasteuri, Streptococcus parasanguinis, and Streptococcus mitis. A significant correlation was observed between the mean proportions of species colonizing BRONJ-like lesions and the teeth of experimental rats (r=0.818, P<0.001). Significant differences were seen in several species colonizing the teeth of control rats compared to experimental rats (P<0.05). Micro-computed tomography analyses revealed higher residual bone in mandibular (P=0.001) and maxillary (P=0.108) tooth sockets of experimental rats. BRONJ-like lesions were colonized mainly by non-pathogenic bacteria. ZA+DX administered to rats at doses equivalent to those given to cancer patients resulted in changes to the oral biofilm and impaired bone healing following tooth extraction.

Photodynamic Inactivation of Actinomyces naeslundii in Comparison With Chlorhexidine and Polyhexanide--A New Approach for Antiseptic Treatment of Medication-Related Osteonecrosis of the Jaw?

PURPOSE: Local antimicrobial therapy is a fundamental principle in the treatment of lesions of medication-related osteonecrosis of the jaw. Antimicrobial photodynamic therapy (aPDT) as a local application for the treatment of microbial infections has become more widely used in recent years. In the mouth, the bone surface is in constant contact with saliva and thus cannot be kept sterile, making the development of strategies for disinfection even more important. Different methods currently in use include local rinses with chlorhexidine (CHX), polyhexanide (PHX), or aPDT. This study compared the efficiency of these 3 methods. MATERIALS AND METHODS: The in vitro activity of 3 different agents against slowly growing Actinomyces naeslundii isolated from a patient with osteonecrosis was evaluated. PHX 0.04% solution, CHX 0.12% solution, and methylene blue (MB) based dye with a laser light of 660-nm wavelength (aPDT) were compared. RESULTS: The decrease in colony-forming units by each method was measured using an in vitro killing assay based on a water-exposed surface in a well plate. MB dye with laser (10 seconds) decreased the bacterial load by more than 4 orders of magnitude and was superior to PHX and CHX exposure for 60 seconds. CONCLUSION: Laser exposure alone and MB dye exposure alone decreased bacterial loads slightly, but less efficiently than 60-second exposure to PHX or CHX. The most effective means of decreasing colony-forming units was achieved by a combination of laser light and dye, which also can be used clinically.

Impact of Actinomyces naeslundii on bisphosphonate-related osteonecrosis of the jaws in ovariectomized rats with periodontitis.

Bisphosphonates-related osteonecrosis of the jaws (BRONJ) is a severe complication of BPs therapy with unknown pathogenesis. This study aimed to evaluate the impact of Actinomyces naeslundii (A. naeslundii) on the progression of BRONJ in ovariectomized (OVX) rat model with periodontal diseases. Sixty rats were randomly assigned into four groups. All rats underwent bilateral ovariectomy. Six weeks after surgery, animals with periodontitis induced by ligature placement were administrated with normal saline (NS), NS &A. naeslundii inoculation, zolecdronic acid (ZA) and ZA &A. naeslundii inoculation for 12 weeks, respectively. Loads of total bacteria and A. naeslundii in the mouth were assessed by real time PCR. After sacrifice, the mandibles were harvested for micro-computed tomography (micro-CT) and histological examination. Real-time PCR demonstrated that A. naeslundii was not routinely found in the rats and ZA treatment did not promote its accumulation. Micro-CT examination disclosed that ligature placement induced significant alveolar bone loss, which was greatly attenuated by ZA treatment and aggravated by A. naeslundii. Histological assessment demonstrated that ZA treatment increased the risk of developing BRONJ-like disease but this condition was not worsen with the presence of A. naeslundii. Our study suggested that oral A. naeslundii inoculation aggravated periodontal disease but not BRONJ in our animal model.

Systemic immunity shapes the oral microbiome and susceptibility to bisphosphonate-associated osteonecrosis of the jaw.

BACKGROUND: Osteonecrosis of the jaw (ONJ) is a rare but serious adverse drug effect linked to long-term and/or high-dose exposure to nitrogen-bisphosphonates (N-BP), the standard of care for the treatment of bone fragility disorders. The mechanism leading to bisphosphonate-associated ONJ (BAONJ) is unclear and optimal treatment strategies are lacking. Recent evidence suggests that BAONJ may be linked to drug-induced immune dysfunction, possibly associated with increased susceptibility to infections in the oral cavity. The objective of this investigation was to comprehensively assess the relationship linking immune function, N-BP exposure, the oral microbiome and ONJ susceptibility. METHODS: Leukocyte gene expression of factors important for immunity, wound healing and barrier function were assessed by real-time quantitative PCR and the oral microbiome was characterized by 454 pyrosequencing of the 16S rRNA gene in 93 subjects stratified by N-BP exposure and a history of ONJ. RESULTS: There were marked differences in the systemic expression of genes regulating immune and barrier functions including RANK (p = 0.007), aryl hydrocarbon receptor (AHR, p < 0.001), and FGF9 (p < 0.001), which were collectively up-regulated in individuals exposed to N-BP without ONJ relative to treatment controls. In contrast, the expression levels of these same genes were significantly down-regulated in those who had experienced BAONJ. Surprisingly, the oral microbiome composition was not directly linked to either BAONJ or N-BP exposure, rather the systemic leukocyte expression levels of RANK, TNFA and AHR each explained 9% (p = 0.04), 12% (p = 0.01), and 7% (p = 0.03) of the oral bacterial beta diversity. CONCLUSIONS: The oral microbiome is unlikely causative of ONJ, rather individuals with BAONJ lacked immune resiliency which impaired their capacity to respond adequately to the immunological stress of N-BP treatment. This may be the common factor linking N-BP and anti-RANK agents to ONJ in at-risk individuals. Preventive and/or therapeutic strategies should target the wound healing deficits present in those with ONJ.

Infection and medication-related osteonecrosis of the jaw.

Medication-related osteonecrosis of the jaw (MRONJ), although initially believed to be exclusively associated with bisphosphonates, has been implicated in recent reports with additional drugs, especially the bone antiresorptive denosumab. The pathophysiology has not been fully elucidated, and no causal association between bone antiresorptive regimens and MRONJ has yet been established. However, reduced bone turnover and infection, an almost universal finding, are thought to be central to the pathogenesis of MRONJ. Both bisphosphonates and denosumab, through different pathways of action, significantly reduce the rate of bone turnover and potentially reduce the efficacy of the host defense against infection. Recent evidence questions the simplified etiology of low bone turnover causing MRONJ and offers evidence on the prominent role of infection instead. The management of MRONJ remains a significant clinical challenge, with little progress having been made on treatment. The aim of this article is to explore the current theories on the etiology of MRONJ and to emphasize the importance of infection in the development of this devastating pathology.

Microbiological investigation of medication-related osteonecrosis of the jaw: preliminary results.

Medication-related osteonecrosis of the jaw (MRONJ) is a well-recognized severe complication of bisphosphonate (BPs) treatment in patients with osteoporosis or metastatic cancer. Microbiological infection has been hypothesized as a contributing factor to bisphosphonate related osteonecrosis of the jaw (BRONJ). Despite infection being present in BRONJ patients, there is no clear data as to whether infection plays a role in the pathophysiology. Moreover, microbial cultures have not been helpful in directing therapy because specific pathogens have not been identified. The objective of this study was to determine the bacterial colonization of jawbone and identify the bacterial phylotypes associated with BRONJ. Twenty oncologic patients, aged 48-87 years (average age 70.65 ± 8.86 years) with BRONJ were enrolled in this study and underwent three different microbiological samplings. Overall, 60 samples were obtained from oral mucosa, necrotic bone fragments and fistula drainage. The same procedure was performed for the laboratory culture of all these specimens. No significant differences regarding either gram+ and gram– species (Chi-squared= 0.1642; p = 0.6854) or aerobes and anaerobes bacteria (Chi-squared= 3.084; p = 0.0791) were found. Compared to other sampling techniques, the oral swab allowed to obtain valuable microbial data in order to recognize pathogens responsible for the infection and to outline a focused antimicrobial therapy.

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Important aspects regarding the role of microorganisms in bisphosphonate-related osteonecrosis of the jaws.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is an important side effect of bisphosphonates, whose etiopathogenesis has not been completely elucidated. Theories pointing to bone turnover and angiogenesis inhibition, as well as effects on epithelial cells of oral mucosa and the role of microorganisms have been reported. Nevertheless, the true contribution of each one of these factors to BRONJ is unknown. We present here a literature review focusing on important aspects regarding the role of microorganisms in BRONJ development. Knowledge about specific microbiota and its role in the etiopathogenesis of this disease can help the optimisation of preventive and therapeutic interventions in patients with or at-risk for BRONJ.

Histomorphologic characteristics of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a known side effect of the therapy with bisphosphonates. No specific pathologic aspects or histological features are included in the most current definition. This study investigates characteristics of BRONJ with a special emphasis on histomorphologic aspects, evaluating the role of Actinomyces spp. as well as other disease-promoting factors in a formal pathogenetic context. METHODS: We investigated 23 patients (14 female, nine male; mean age: 66 ± 11.8 years) who received bisphosphonates with a gender- /age-matched control group. Tissue specimens were treated according to local standards and analyzed histologically. RESULTS: In 18 (78.3%) BRONJ cases, we found Actinomyces spp. colonies. Bone remodeling could be found in three specimens (13%). Eight specimens (34.8%) showed signs of epithelial proliferation. Analysis of dental treatment before the onset of BRONJ did not reveal significant differences (P > 0.20). In 10 patients (83%; P > 0.05) of the reported cases a relationship between dental treatment and the occurrence of a purulent bone necrosis could be observed. Statistically significant differences in thickness of trabeculae were detected between the two study groups (P = 0.04). CONCLUSIONS: This study demonstrates the important influence of the osteoblast-osteoclast balance in a histomorphologic analysis. Together with cofactors, which are able to trigger the onset of BRONJ, a new pathogenesis model was developed.

Analysis of the factors affecting the formation of the microbiome associated with chronic osteomyelitis of the jaw.

Chronic osteomyelitis of the jaw (COMJ) is one of the most intractable diseases among head and neck infections. Antimicrobial agents are routinely administered for COMJ without sufficient bacterial information, resulting in frequent treatment failures. To improve our knowledge of the bacterial aetiology of COMJ and to assist in the development of effective treatments, we performed a comprehensive analysis of the microbiome. Sixteen patients with four clinical types of COMJ (four with suppurative osteomyelitis, three with osteoradionecrosis of the jaw, four with primary chronic osteomyelitis, and five with bisphosphonate-related osteonecrosis of the jaw) were enrolled in this study. Bone samples were subjected to bacterial community comparisons by 16S rRNA gene pyrosequencing. As a result, we clarified that COMJ was caused by a far greater range of bacterial species (12 phyla and 163 genera) than previously reported. Moreover, the bacterial structures in COMJ changed dramatically with disease stage and the condition of the affected bone. Multiple correlation analyses revealed that sequestration and bone exposure could affect the community structure. On the basis of these factors, we reclassified COMJ into three clinical stages: I, inflamed or sclerotic bone without exposure; II, sequestrum without exposure; and III, exposed sequestrum. In stage II, the bacterial diversity was significantly lower, and the anaerobe genera Fusobacterium, Tannerella (formerly Bacteroides) and Porphyromonas were more abundant, than observed during other stages. Because these bacteria habitually reside in any clinical stage, they were considered to constitute the core microbiome of COMJ. Targeting these bacteria should lead to the development of effective preventive measures and cures.

Bacterial promotion of bisphosphonate-induced osteonecrosis in Wistar rats.

Clinically, bisphosphonate-related osteonecrosis (BRON) only occurs in the jaw (BRONJ). We aimed to determine differences between the jaw and other bones, as well as the relationship between periodontal pathogens and BRON. Twelve male Wistar rats were divided into two groups: group Z (n=6) were subcutaneously injected with zoledronic acid weekly for 4 weeks; group C (n=6) were injected with saline. One week after the final injection, rats in both groups were divided into three subgroups: Aa subgroup was injected with freeze-dried Aggregatibacter actinomycetemcomitans into bone marrow of the mandibles and femurs, while CFA and saline subgroups were injected with complete Freund's adjuvant (CFA) or saline using the same protocol. Four weeks after those injections, the rats were euthanized. Areas of osteonecrosis were measured histologically. Statistically, rats in group Z showed significantly wider osteonecrosis areas in both mandibles and femurs with each type of local injection than rats in group C. In group Z, mandibles and femurs stimulated with A. actinomycetemcomitans or CFA had significantly wider osteonecrosis areas than those stimulated with saline. We have developed a rat model with BRONJ-like lesions. Our results showed BRON to develop not only in the jaw but also in other types of bone following an inflammatory stimulus.